Discovery of a somatic mutation in myd88 gene in lymphoplasmacytic lymphoma

ABSTRACT

Diagnostic assays for facilitating the diagnosis of lymphoplasmacytic lymphoma (LPL) are provided. The method comprises assessing a biological sample of the subject for the presence of a mutation at position 38182641 in chromosome 3p22.2, wherein presence of the mutation is indicative that the subject has LPL. Also, provided are targeted therapies, methods for monitoring the progression or recurrence of LPL, and a sensitive and inexpensive real-time allele specific polymerase chain reaction assay for reliable and quantitative assessments of the mutation.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/128,241, filed Mar. 18, 2014 which is a national stage filing under35 U.S.C. § 371 of International Application PCT/US2012/044956, filedJun. 29, 2012, which was published under PCT Article 21(2) in English,and which claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 61/571,657, filed Jul. 1, 2011, thedisclosure of each referenced application is incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

Waldenstrom's macroglobulinemia (WM) is a distinct clinicopathologicalentity resulting from the accumulation, predominantly in the bonemarrow, of clonally related lymphoplasmacytic cells which secrete amonoclonal IgM protein. This condition is considered to correspond tolymphoplasmacytic lymphoma (LPL) as defined by the World HealthOrganization classification system. Genetic factors play an importantrole in the pathogenesis of WM, with 25% of patients demonstrating afamily history. IgM monoclonal gammopathy of unknown significance (MGUS)often precedes the development of WM. The primary oncogenetic eventresulting in malignant transformation in WM remains to be delineated.Knowledge of such genomic alteration(s) may permit advances indiagnostic testing, and development of targeted therapies.

SUMMARY OF THE INVENTION

It has been discovered, surprisingly, that a somatic mutation in themyeloid differentiation primary response (MYD88) gene is associated withlymphoplasmacytic lymphoma. Accordingly, in some aspects, the inventioninvolves, facilitating the diagnosis of lymphoplasmacytic lymphoma in asubject by selecting a subject on the basis that the subject presentsone or more of the following clinical features: anemia, hyper-viscosity,neuropathy, coagulopathies, splenomegaly, hepatomegaly, adenopathy, andan IgM serum paraprotein, obtaining a biological sample of the subject,and determining from the biological sample whether the subject has amutation at position 38182641 in chromosome 3p22.2, wherein the presenceof the mutation is indicative that the subject has lymphoplasmacyticlymphoma. In some embodiments, the subject presents two or more of theclinical features. In some embodiments, the subject presents three ormore of the clinical features. In some embodiments, said determiningcomprises performing an assay to interrogate position 38182641 inchromosome 3p22.2. In some embodiments, the assay comprises allelespecific polymerase chain reaction performed using an allele specificprimer, wherein the allele specific primer hybridizes at or near its 3′end to the mutation at position 38182641 in chromosome 3p22.2. In someembodiments, the allele specific primer is SEQ ID NO: 5.

According to one aspect of the invention, a method to distinguishlymphoplasmacytic lymphoma from other B cell neoplasms is provided. Themethod comprises obtaining a biological sample from a subject thatpresents symptoms of a B cell lymphoma, which symptom does not exclude adiagnosis of lymphoplasmacytic lymphoma. Symptoms of a B cell lymphomawhich do not exclude a diagnosis of LPL include asymptomatic localizedor generalized peripheral lymphadenopathy, plasmacytic difference, bonemarrow involvement, autoimmune thrombocytopenia, end organ damage (renalinsufficiency), anemia, hyper-viscosity, neuropathy, coagulopathies,splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein. Insome embodiments, said determining comprises performing an assay tointerrogate position 38182641 in chromosome 3p22.2. In some embodiments,the assay comprises allele specific polymerase chain reaction performedusing an allele specific primer, wherein the allele specific primerhybridizes at or near its 3′ end to the mutation at position 38182641 inchromosome 3p22.2. In some embodiments, the allele specific primer isSEQ ID NO: 5.

In some embodiments, the method comprises obtaining a biological samplefrom a subject that presents symptoms of both LPL and at least one Bcell neoplasm selected from the group consisting of nodal marginal zonelymphomas, extranodal marginal zone lymphoma of mucosa-associatedlymphoid tissue (MALT lymphoma), splenic B cell marginal zone lymphoma,monoclonal gammopathy of undetermined significance and plasma cellmyeloma, determining from the biological sample whether the subject hasa mutation at position 38182641 in chromosome 3p22.2, and providing areport whether the subject has a mutation at position 38182641 inchromosome 3p22.2, wherein the presence of the mutation is indicativethat the subject has lymphoplasmacytic lymphoma.

According to one aspect of the invention, a method to treatlymphoplasmacytic lymphoma in a subject is provided. The methodcomprises selecting a subject on the basis that the subject has amutation at position 38182641 in chromosome 3p22.2. In some embodiments,the subject also presents one or more symptoms or clinical features ofLPL, such as, anemia, hyper-viscosity, neuropathy, coagulopathies,splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein.The subject is administered a myeloid differentiation primary response88 (MYD88) inhibitor, an interleukin receptor associate kinase 1/4(IRAK-1/4) inhibitor, and/or a Bruton's tyrosine kinase (BTK) inhibitorin an amount effective to treat lymphoplasmacytic lymphoma. In someembodiments, the MYD88 inhibitor is a peptidomimetic compound ST2825. Insome embodiments, the IRAK-1/4 inhibitor isN-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole. In someembodiments, the BTK inhibitor is Ibrutinib (PCI-32765).

According to one aspect of the invention, a method for monitoringprogression or recurrence of lymphoplasmacytic lymphoma in a subject isprovided. The method comprises obtaining multiple biological samples ofa subject over a period of time, determining from the multiplebiological samples the level of a transcript comprising a mutation atposition 38182641 in chromosome 3p22.2, wherein a change in the level ofthe transcript over the period of time is indicative of the progressionor recurrence of LPL in the subject. In some embodiments, the subject isundergoing chemotherapy to treat LPL. In some embodiments, the level ofthe transcript is measured using quantitative real time polymerase chainreaction. In some embodiments, the quantitative real time polymerasechain reaction is performed using an allele specific primer, wherein theallele specific primer hybridizes at or near its 3′ end to the mutationat position 38182641 in chromosome 3p22.2. In some embodiments, theallele specific primer is SEQ ID NO: 5.

According to one aspect of the invention, a method for detecting amutation at position 38182641 in chromosome 3p22.2 in a subject isprovided. The method comprises obtaining a biological sample from thesubject in need of such detection, determining from the biologicalsample whether the subject has a mutation at position 38182641 inchromosome 3p22.2 by allele specific polymerase chain reaction performedusing an allele specific primer wherein the allele specific primerhybridizes at or near its 3′ end to the mutation at position 38182641 inchromosome 3p22.2.

According to one aspect of the invention, a method for facilitating thediagnosis of lymphoplasmacytic lymphoma in a subject is provided. Themethod comprises selecting a subject on the basis that the subjectpresents one or more of the following clinical features: anemia,hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly,adenopathy, and an IgM serum paraprotein, obtaining a biological sampleof the subject, determining from the biological sample whether thesubject has a mutation at position 38182641 in chromosome 3p22.2, andproviding a report summarizing statistically significant resultsindicating that the subject has lymphoplasmacytic lymphoma if thesubject has the mutation.

The biological sample includes, but is not limited to, a sample of bonemarrow, lymph node, spleen or blood. In some embodiments, the mutationresults in a single nucleotide change from T to C in the myeloiddifferentiation primary response 88 (MYD88) gene. In some embodiments,the mutation results in an amino acid change from leucine to proline atposition 265 in the myeloid differentiation primary response 88 protein.

The subject (individual) is a human. In some embodiments, the subject issuspected of LPL and presents one or more of the clinical features ofLPL.

Without intending to be bound by the theory of the invention, it isbelieved that the presence of the mutation in a subject at position38182641 in chromosome 3p22.2 is predictive of the risk of developmentof LPL. The presence of the mutation at position 38182641 in chromosome3p22.2 in a subject having an elevated (abnormal) level of monoclonalIgM serum paraprotein is predictive of the risk of development of LPL.In some embodiments, a method for predicting the risk of development ofLPL is provided. The method comprises selecting a subject on the basisthat the subject has an elevated (abnormal) level of monoclonal IgMserum paraprotein, and determining whether the subject has a mutation atposition 38182641 in chromosome 3p22.2, wherein the presence of themutation is indicative that the subject is at an increased risk ofdeveloping LPL.

It may even be that the few instances where the mutation was present ina subject who is diagnosed with a B cell neoplasm (other than LPL andsubtype ABC of diffuse large B cell lymphoma) was a mistaken diagnosis.

These and other aspects of the inventions, as well as various advantagesand utilities will be apparent with reference to the DetailedDescription. Each aspect of the invention can encompass variousembodiments as will be understood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show acquired uniparental disomy (aUPD) at chromosome 3pencompassing MYD88 as determined by WGS. Chromosome wide datademonstrating copy number neutral loss of heterozygosity withcorresponding allele imbalance for patients WM1 and WM3 indicatingpresence of an aUPD throughout the tumor clone (FIG. 1A). For patientsWM2 and WM4, an aUPD was present in a subpopulation of tumor cells asdemonstrated by sustained allele imbalance. Confirmatory Sangersequencing of the MYD88 mutation mirrors allele imbalances observed byWGS (FIG. 1B). Chromosome wide data (FIG. 1C) and confirmatory Sangersequencing for MYD88 L265 (FIG. 1D) using CD19-depleted PB mononuclearcells for normal tissue comparisons in patients WM1 and WM2. Arrows(FIGS. 1B,1D) denote variant allele position.

FIGS. 2A-2D show agarose gel-based AS-PCR assay for detection of MYD88L265P. The reverse primers with an internal mismatch in the thirdposition from the 3′-end and the common forward primer were indicated bythe arrows (FIG. 2A). Sanger sequencing confirmed that DLBCL cell lineOCI-LY3 carried homozygous MYD88 L265P, whereas OCI-LY19 was wild-typefor MYD88 L265P. The position of L265P was indicated by arrow (FIG. 2B).Sensitivity of the AS-PCR assay was assessed by a serial dilution of theMYD88 L265P mutant DNA from OCI-LY3 with the wild-type DNA fromOCI-LY19. The PCR products (159-bp) were separated on 2% agarose gel andindicated by arrows (FIG. 2C). The mutant MYD88 L265P allele canconsistently be detected at a dilution of 0.1%. An example of WMpatients who carried homozygous MYD88 L265P. This patient had 70% BMinvolvement (FIG. 2D).

FIGS. 3A-3D show the sensitivity and specificity of the real-time AS-PCRand scatter plot of comparison of MYD88 L265P positive and negativepatients and healthy donors. The MYD88 L265P mutant DNA (OCI-LY3) wasdiluted with the wild-type DNA (OCI-LY19) at the concentration asindicated in the amplification plot. The mutant MYD88 L265P allele canbe detected at a dilution of 0.08% (FIG. 3A). Correlation coefficient ofthe standard curve was 0.998 with a slope value of −3.48 (FIG. 3B).Melting curve analysis revealed that the MYD88 L265P mutant-specificamplicon melted at 84° C. A minor non-specific amplification was onlyfound in the dilution of 0.4% or lower with a melting peak at 80° C.(FIG. 3C). Scatter plot showed two major clusters of WM patients thatwere separated by 3.2 cycles. The cluster with delta C_(T) valuesranging from 9.6 to 15.2 cycles was similar to healthy donors (deltaC_(T) values ranging from 10.7 to 16.9 cycles). This cluster wasdetermined as MYD88 L265P negative, whereas another cluster with deltaC_(T) values ranging from −0.2 to 6.4 cycles was determined as MYD88L265P positive (FIG. 3D).

FIG. 4 shows increased phosphorylation of BTK by western blotting withphospho-specific antibody in Waldenstrom's Macroglobulinemia (WM) celllines, BCWM.1 and MWCL-1, compared to Multiple myeloma cell lines, ANBL6and INA6. Antibody against total BTK was used as loading control.PCI-32765 significantly blocked the BTK phosphorylation in WM cells.

FIG. 5 shows that PCI-32765 blocked the downstream NF-kB, MAPK, Stat3signaling by significantly reducing the phosphorylation of IKBα, ERK1/2and Stat3 proteins in WM cell lines, BCWM.1 and MWCL-1, compared tomultiple myeloma cell lines, ANBL6 and INA6. Antibodies againstcorresponding total proteins and GAPDH were used as loading controls.

FIG. 6 shows that knockdown of MYD88 by lentiviral transduction, and/oruse of a MYD88 inhibitor leads to decreased BTK phosphorylation.Antibodies against total BTK and/or GAPDH were used as loading control.

FIGS. 7A-7C demonstrate that BTK inhibitor PCI-32765 induces apoptosisof MYD88 L265P expressing WM cells alone and in combination with MYD88pathway inhibitor and IRAK 1/4 kinase inhibitor. Apoptosis analysis wasperformed using Annexin V and PI staining after PCI-32765 treatment for24 hrs (FIG. 7A). In FIG. 7B, apoptosis analysis was performed usingannexin V and PI staining after PCI-32765 and MYD88 homodimerizationinhibitor treatment for 24 hrs. 1: DMSO; 2: PCI-32765 (1.0 μM); 3: MYD88inhibitory peptides (100 μM); 4: PCI-32765+MYD88 inhibitory peptides.PCI-32765 shows synergistic tumor cell killing in combination with anIRAK 1/4 kinase inhibitor (FIG. 7C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in one aspect, relates to the surprisingdiscovery of a somatic mutation in the myeloid differentiation primaryresponse (MYD88) gene in patients with lymphoplasmacytic lymphoma. Inparticular, the invention is based on the identification of a somaticmutation at position 38182641 in chromosome 3p22.2 which results in asingle nucleotide change from T→C in the myeloid differentiation primaryresponse (MYD88) gene, and a predicted non-synonymous change at aminoacid position 265 from leucine to proline (L265P). While previous workhas identified the same mutation in subtype ABC of diffuse large B celllymphoma, this previous work did not make any association between themutation and lymphoplasmacytic lymphoma.

Since the molecular mechanism of LPL was unknown, the differentialdiagnosis of many diseases that are morphologically similar to LPL washampered. The discovery set forth in the instant application helps todiscriminate LPL from other overlapping entities, and allows for diseasespecific treatment targeting.

According to one aspect, the present invention provides diagnosticassays for facilitating or aiding in the diagnosis of lymphoplasmacyticlymphoma (LPL) in a subject. LPL is a neoplasm of small B lymphocytes,plasma cytoid lymphocytes, and plasma cells, usually involving bonemarrow (BM) and sometime lymph nodes and spleen. Waldenstrommacroglobulinemia (WM) is found in a significant subset of patients withLPL and is defined as LPL with BM involvement and an IgM monoclonalgammopathy of any concentration.

According to one aspect of the invention, a subject is selected forassessment of a mutation at position 38182641 in chromosome 3p22.2 onthe basis that the subject is suspected of having LPL, and a biologicalsample of the subject is assessed for the presence of a mutation. Thepresence of the mutation is indicative that the subject has LPL. As usedherein, “selecting a subject” means identifying a subject that presentsone or more clinical features of LPL for further diagnostic analysis.The one or more clinical features of LPL include anemia,hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly,adenopathy, and an IgM serum paraprotein. In some embodiments, a subjectpresenting two or more, three or more, four or more, five or more, sixor more, or seven or more of these clinical features is selected. Thesubject is selected by a medical practitioner (e.g., a doctor, nurse,clinical laboratory practitioner, genetic counselor, etc.), a healthcareorganization, or a clinical laboratory.

Non-limiting examples of the biological sample include bone marrow,lymph node, spleen or blood. Obtaining a biological sample of a subjectmeans taking possession of a biological sample of the subject. Obtaininga biological sample from a subject means removing a biological samplefrom the subject. Therefore, the person obtaining a biological sample ofa subject and determining the presence of the mutation in the sampledoes not necessarily obtain the biological sample from the subject. Insome embodiments, the biological sample may be removed from the subjectby a medical practitioner (e.g., a doctor, nurse, or a clinicallaboratory practitioner), and then provided to the person determiningthe presence of the mutation. The biological sample may be provided tothe person determining the mutation by the subject or by a medicalpractitioner (e.g., a doctor, nurse, or a clinical laboratorypractitioner). In some embodiments, the person determining the mutationobtains a biological sample from the subject by removing the sample fromthe subject.

The term “mutation” means any change or difference in the nucleic acidor protein sequence of MYD88 as compared to the wild type sequence thatresults in the activation of MYD88 which leads to the activation ofNF-κB. Mutations include, but are not limited to, nonsense mutations,missense mutations, frameshift mutations, rearrangement mutations,insertion mutations and deletion mutations. In some embodiments, themutation is a somatic mutation at position 38182641 in chromosome 3p22.2which results in a single nucleotide change from T→C in the myeloiddifferentiation primary response (MYD88) gene, and a predictednon-synonymous change at amino acid position 265 from leucine to proline(L265P).

One skilled in the art will appreciate that many suitable methods, inaddition to and including the ones discussed in the examples, can beused to detect mutations in the MYD88 gene. Detection methods that canbe used include, but are not limited to, direct sequencing, DNAchiptechnologies, mass spectroscopy, polymerase chain reaction (PCR), allelespecific polymerase chain reaction, real time polymerase chain reaction,reverse transcriptase PCR, electrophoretic mobility, nucleic acidhybridization, fluorescent in situ hybridization, and denaturing highperformance liquid chromatography.

In some embodiments, mutations in the MYD88 gene may be detected byallele specific polymerase chain reaction (AS-PCR). For AS-PCR, allelespecific primers are used which hybridize at or near their 3′ ends to aparticular mutation in the MYD88 gene. If the mutation is not present,the 3′-terminal mismatched primer does not initiate replication, and anamplification product is not observed. In some embodiments, only theforward primer or the reverse primer hybridizes at or near its 3′ endsto a particular mutation in the MYD88 gene. In some embodiments, boththe forward and the reverse primer hybridize at or near their 3′ ends toa particular mutation in the MYD88 gene. In some embodiments, the allelespecific primer is SEQ ID NO: 5. In some embodiments, the mutation is asomatic mutation at position 38182641 in chromosome 3p22.2 which resultsin a single nucleotide change from T→C in the myeloid differentiationprimary response (MYD88) gene, and a predicted non-synonymous change atamino acid position 265 from leucine to proline (L265P).

In some embodiments, mutations in the MYD88 gene may be detected by thedirect sequencing of nucleic acid molecules. Techniques for the directsequencing of DNA are well known in the art. In one embodiment of theinvention, mutations may be detected by Sanger sequencing, which mayinclude the use of a thermostable polymerase enzyme, a sequencingprimer, dNTPs and limiting amounts of chain terminating fluorescently orradioactively labeled ddNTPs. Polyacrylamide gel electrophoresis oranother technique such as capillary electrophoresis may be used toseparate the products of the sequencing reactions followed by thedetection of the fluorescent or radioactive labels. In one example ofthis embodiment of the invention, mutations in MYD88 could be determinedusing automated sequencing on an Applied Biosystems 3700 DNA Analyzer or3730×1 DNA Analyzer™. Mutations may be identified by comparing thesequence of a subject to that of a wildtype individual or to referencesequences found in the public databases.

Other embodiments of the invention contemplate the use of DNA chiptechnologies for the detection of mutations within the MYD88 gene. Amongother applications, DNA chip technologies allow for the identificationof mutations within the sequences of the intention through the analysisof the hybridization patterns of a nucleic acid sample onto ahigh-density spatially addressable microarray of predeterminedsequences.

Another technique for the detection of mutations is denaturing HPLCanalysis. Accordingly, one embodiment of the invention includes the useof a Transgenomic Wave™ machine for the dHPLC analysis of nucleic acidsfor the identification of heterozygous mutations or polymorphisms withinthe sequences of the invention.

The invention also contemplates the use of mass spectroscopy for thegenotyping of mutations. Mutant and wildtype nucleic acid molecules maydiffer in mass due to the different composition of wildtype and mutantsequences, allowing for the identification of mutations on the basis ofthe molecular mass of different nucleotide sequences. The use of massspectroscopy, and in particular Matrix Assisted Laser DesorptionIonisation Time of Flight (MALDI-TOF) mass spectroscopy for thegenotyping of mutations is well known by those skilled in the relevantart. For example, U.S. Pat. No. 6,043,031 describes a fast and highlyaccurate mass spectrometer based process for detecting a particularnucleic acid sequence. The MassARRAY™ platform from SEQUENOM™ is anexample of a commercially available system capable of genotyping singlenucleotide polymorphisms and detecting the mutations in genes.

According to one aspect, the present invention provides a method todistinguish lymphoplasmacytic lymphoma from other B cell neoplasmsselected from the group consisting of nodal marginal zone lymphomas,extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue(MALT lymphoma), splenic B cell marginal zone lymphoma, monoclonalgammopathy of undetermined significance and plasma cell myeloma. Themethod comprises selecting or identifying a subject that presents one ormore symptoms or clinical features of LPL which overlap with one or moresymptoms of at least one of the B cell neoplasms described above. Thus,the subject is an individual who is suspected of having either LPL orone of the other B cell neoplasm. The subject is selected for furtherdiagnostic analysis by a medical practitioner (e.g., a doctor, nurse,clinical laboratory practitioner, genetic counselor, etc.), a healthcareorganization, or a clinical laboratory.

The one or more symptoms or clinical features of LPL include anemia,hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly,adenopathy, and an IgM serum paraprotein. In addition, the subject mayalso present one or more of the following clinical features or symptomsof other B cell neoplasms: asymptomatic localized or generalizedperipheral lymphadenopathy, plasmacytic difference, bone marrowinvolvement, autoimmune thrombocytopenia, peripheral blood villouslymphocytes, end organ damage (hypercalcemia, renal insufficiency, bonelesions), recurrent infections, elevated creatine, hyperuricemia, andhypoalbunemia. The subject suspected of having either LPL or one of theother B cell neoplasm is assessed for the presence of a mutation atposition 38182641 in chromosome 3p22.2, wherein the presence of themutation is indicative that the subject has LPL.

A report summarizing the results of the analysis, i.e. the presence orabsence of the mutation and any other information pertaining to theanalysis could optionally be generated as part of the analysis (whichmay be interchangeably referred to herein as “providing” a report,“producing” a report, or “generating” a report). Examples of reports mayinclude, but are not limited to, reports in paper (such ascomputer-generated printouts of test results) or equivalent formats andreports stored on computer readable medium (such as a CD, computer harddrive, or computer network server, etc.). Reports, particularly thosestored on computer readable medium, can be part of a database (such as adatabase of patient records, which may be a “secure database” that hassecurity features that limit access to the report, such as to allow onlythe patient and the patient's medical practitioners to view the report,for example). In addition to, or as an alternative to, generating atangible report, reports can also be displayed on a computer screen (orthe display of another electronic device or instrument).

A report can further be transmitted, communicated or reported (theseterms may be used herein interchangeably), such as to the individual whowas tested, a medical practitioner (e.g., a doctor, nurse, clinicallaboratory practitioner, genetic counselor, etc.), a healthcareorganization, a clinical laboratory, and/or any other party intended toview or possess the report. The act of ‘transmitting’ or ‘communicating’a report can be by any means known in the art, based on the form of thereport, and includes both oral and non-oral transmission. Furthermore,“transmitting” or “communicating” a report can include delivering areport (“pushing”) and/or retrieving (“pulling”) a report. For example,non-oral reports can be transmitted/communicated by such means as beingphysically transferred between parties (such as for reports in paperformat), such as by being physically delivered from one party toanother, or by being transmitted electronically or in signal form (e.g.,via e-mail or over the internet, by facsimile, and/or by any wired orwireless communication methods known in the art), such as by beingretrieved from a database stored on a computer network server, etc.

According to one aspect of the invention, a method to treat LPL isprovided. The method comprises selecting a subject on the basis that thesubject has a mutation at position 38182641 in chromosome 3p22.2, andadministering to the subject a myeloid differentiation primary response88 (MYD88) inhibitor, an interleukin receptor associate kinase 1/4(IRAK-1/4) inhibitor, and/or a Bruton's tyrosine kinase (BTK) inhibitorin an amount effective to treat lymphoplasmacytic lymphoma. Anon-limiting example of an MYD88 inhibitor includes the peptidomimeticcompound ST2825 (WO 2006/06709). A non-limiting example of an IRAK-1/4inhibitor isN-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole. In someembodiments, BTK inhibitors useful in the instant invention block MYD88L265P and BTK signaling. A non-limiting example of a BTK inhibitorincludes Ibrutinib (PCI-32765).

In some embodiments, the method comprises selecting a subject on thebasis that the subject has a mutation at position 38182641 in chromosome3p22.2 and presents one or more symptoms of LPL. The selected subject istreated using an effective amount of bortezomib (Velcade®),bendamestine, alemtuzumab, and/or rituximab, but not bisphosphonates orfludarabine.

The MYD88 inhibitor, IRAK-1/4 inhibitor, and/or BTK inhibitor areadministered in an effective amount. An effective amount is a dosesufficient to provide a medically desirable result and can be determinedby one of skill in the art using routine methods. In some embodiments,an effective amount is an amount which results in any improvement in thecondition being treated. In some embodiments, an effective amount maydepend on the type and extent of LPL being treated and/or use of one ormore additional therapeutic agents. However, one of skill in the art candetermine appropriate doses and ranges of therapeutic agents to use, forexample based on in vitro and/or in vivo testing and/or other knowledgeof compound dosages.

When administered to a subject, effective amounts of the therapeuticagent will depend, of course, on the particular disease being treated;the severity of the disease; individual patient parameters includingage, physical condition, size and weight, concurrent treatment,frequency of treatment, and the mode of administration. These factorsare well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. In someembodiments, a maximum dose is used, that is, the highest safe doseaccording to sound medical judgment.

In the treatment of LPL, an effective amount is that amount which slowsthe progression of the disease, halts the progression of the disease, orreverses the progression of the disease. An effective amount includes,but is not limited to, that amount necessary to slow, reduce, inhibit,ameliorate or reverse one or more symptoms associated with LPL. In someembodiments, such terms refer to a reduction in the levels of IgM serumparaprotein, anemia, hyper-viscosity, neuropathy, coagulopathies,splenomegaly, hepatomegaly, and adenopathy.

An effective amount of a compound typically will vary from about 0.001mg/kg to about 1000 mg/kg in one or more dose administrations, for oneor several days (depending of course of the mode of administration andthe factors discussed above).

Actual dosage levels of the therapeutic agent can be varied to obtain anamount that is effective to achieve the desired therapeutic response fora particular patient, compositions, and mode of administration. Theselected dosage level depends upon the activity of the particularcompound, the route of administration, the tissue being treated, andprior medical history of the patient being treated. However, it iswithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effort and togradually increase the dosage until the desired effect is achieved.

Pharmaceutical preparations and compounds comprising MYD88 inhibitor,IRAK-1/4 inhibitor, and/or BTK inhibitor are administered to a subjectby any suitable route. For example, compositions can be administeredorally, including sublingually, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically andtransdermally (as by powders, ointments, or drops), bucally, or nasally.The pharmaceutical preparations of the present invention may include orbe diluted into a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible fillers, diluants or other such substances, which aresuitable for administration to a human or other mammal such as a dog,cat, or horse. The term “carrier” denotes an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate the application. The carriers are capable ofbeing commingled with the preparations of the present invention, andwith each other, in a manner such that there is no interaction whichwould substantially impair the desired pharmaceutical efficacy orstability. Carriers suitable for oral, subcutaneous, intravenous,intramuscular, etc. formulations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

According to one aspect of the invention, a method for monitoringprogression or recurrence of lymphoplasmacytic lymphoma in a subject isprovided. The method comprises obtaining multiple biological samples ofa subject over a period of time, determining from the multiplebiological samples the level of a transcript comprising a mutation atposition 38182641 in chromosome 3p22.2, wherein an increase in the levelof the transcript over the period of time is indicative of theprogression or recurrence of LPL in the subject.

As used herein, a transcript comprising a mutation means MYD88 nucleicacid or protein that has a mutation at position 38182641 in chromosome3p22.2. The level of the transcript comprising a mutation at position38182641 in chromosome 3p22.2 can be determined by any means known toone skilled in the art, including, but not limited to Western blot,Northern blot, and quantitative real time polymerase chain reaction. Insome embodiments, quantitation of the transcript levels is accomplishedby quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. Methods of quantitativereal-time PCR are well known in the art. In some embodiments, thequantitative real time polymerase chain reaction is performed using anallele specific primer, wherein the allele specific primer hybridizes ator near its 3′ end to a mutation at position 38182641 in chromosome3p22.2. In some embodiments, the allele specific primer is SEQ ID NO: 5.

The level of the transcript comprising the mutation is indicative of thestate of the disease. In normal (healthy) subjects the mutation isabsent. In diseased subjects, the transcript comprising the mutation isexpressed at a level consistent with the status (progression) of thedisease. Although it is believed that most of the transcription of theMYD88 gene occurs in the bone marrow, levels of the transcript andprotein will be present in the circulation because of the normalturnover and presence of dead cells in the blood.

As used herein, a change in the level of the transcript comprising amutation means that the amount or concentration of the transcriptsufficiently changes over time. A change in the level of the transcriptover the period of time may be any statistically significant changewhich is detectable. Such a change may include, but is not limited to,about a 1%, about a 10%, about a 20%, about a 40%, about a 80%, about a2-fold, about a 4-fold, about an 8-fold, about a 20-fold, or about a100-fold change over the time. An increase in the level of thetranscript indicative of unfavorable progression of the disease, while adecrease in the level of the transcript is indicative of a favorableprogression of the disease.

As used herein, a “period of time” is intended to include a period ofdays, weeks, months or even years. Multiple biological samples of thesubject are obtained over a period of time, i.e. a biological sample isobtained periodically over time at various intervals. A biologicalsample can be obtained at any interval. For example, a biological samplecan be taken every day for weeks, months or years. Alternatively, abiological sample can be obtained once a week, or six times a week for aperiod of weeks, months or years. In one embodiment, a biological sampleis obtained once a week over a period of three months. In oneembodiment, a biological sample is obtained once a month for a period ofmonths, or years.

In some embodiments, the subject is undergoing chemotherapy to treatLPL. An increase in the level of the transcript over the period of timein a subject undergoing chemotherapy to treat LPL would indicate thatthere is progression of the disease and that the subject is notresponding to the therapy. A decrease in the level of the transcriptover the period of time in a subject undergoing chemotherapy to treatLPL would indicate that the subject is responding to the therapy.

According to one aspect of the invention, a method for detecting amutation at position 38182641 in chromosome 3p22.2 in a subject isprovided. The method comprises obtaining a biological sample from thesubject in need of such detection, and determining from the biologicalsample whether the subject has a mutation at position 38182641 inchromosome 3p22.2 by allele specific polymerase chain reaction (AS-PCR).The AS-PCR is performed using an allele specific primer wherein theallele specific primer hybridizes at or near its 3′ end to the mutationat position 38182641 in chromosome 3p22.2. In some embodiments, theallele specific primer is SEQ ID NO: 5. In some embodiments, themutation is a somatic mutation at position 38182641 in chromosome 3p22.2which results in a single nucleotide change from T→C in the myeloiddifferentiation primary response (MYD88) gene, and a predictednon-synonymous change at amino acid position 265 from leucine to proline(L265P).

A subject in need of detection may be a subject suspected of having LPL.The subject may present one or more clinical features of LPL including,but not limited to anemia, hyper-viscosity, neuropathy, coagulopathies,splenomegaly, hepatomegaly, adenopathy, and an IgM serum paraprotein. Insome embodiments, a subject in need of detection may be a subjectsuspected of having subtype ABC of diffuse large B cell lymphoma. Thesubject may present one or more clinical features of subtype ABC ofdiffuse large B cell lymphoma including, but not limited to enlargedlymph node in the neck, groin or abdomen, fever, weight loss, anddrenching night sweats. A subject in need of detection may be a subjectsuspected of having gastric mucosa-associated lymphoid tissue (MALT)lymphoma. The subject may present one or more clinical features of MALTincluding, but not limited to chronic inflammation caused byHelicobacter pylori infection, stomach pain, dyspepsia, nausea,constipation and anemia.

The present invention is further illustrated by the following Example,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1

Materials and Methods

Thirty patients meeting consensus criteria for the diagnosis of WM werestudied by whole genome sequencing (WGS). Their participation wasapproved by the Institutional Review Board at Dana-Farber CancerInstitute. The characteristics of these patients are summarized inTable 1. Nine of these patients had a familial history of a B-cellmalignancy. Bone marrow (BM) and peripheral blood (PB) mononuclear cellsfrom these individuals were obtained after density-gradientcentrifugation. Magnetic bead sorting was used for tumor cell isolation(Miltenyi Biotec, Auburn, Calif.). The purity of isolated B-cells(CD19+) by this technique was over 90%, and the median clonal B-cellpopulation by light chain restriction was 96% (Table 1). The median timefrom diagnosis of WM to BM collection for the 30 patients was 14.1(range 0-138.2 months). CD19-depleted PB mononuclear cells werecollected as matched normal tissue from 19 of the 30 patients, andpaired tumor/normal tissue WGS performed for 10 of these patients. Thebaseline characteristics for these 10 patients did not differsignificantly from unpaired patients.

Following cell isolation, high molecular weight DNA was isolated usingthe Allprep DNA/RNA mini kit (Qiagen, Valencia, Calif.). For 10patients, WGS of tumor and matched normal samples was performed, and for20 additional patients tumor samples alone were completely sequenced.Library construction and WGS of paired-end clones was performed byComplete Genomics Inc. (CGI; Mountain View, Calif.) as previouslydescribed. Read sequences were aligned to the reference genome (NCBIBuild 37) and reads in potentially variant regions (SNVs, insertions,and deletions) were identified and subjected to local de novo assembly.The assembler then scored both variant and homozygous referenceconsensus calls in each sample using a Bayesian framework whichconsidered read depth, individual base call quality, and mappingprobabilities. A likelihood ratio test for each variant (variant score)expressed in decibels (dB) was generated and reported. High confidencesomatic variants were identified using CGAT version 1.3 with a somaticscore of 0.1 giving an estimated rate of one false somatic singlenucleotide variant per 17.7 Mb of DNA. Copy number was estimated by % GCnormalized read depth, and acquired uniparental disomy (aUPD) wasidentified as copy-neutral loss of heterozygosity. In addition alleleimbalance was determined by the percentage of reads mapping to the minorallele at heterozygous SNPs which was averaged over 500 Kb.

To validate WGS results, a set of PCR primers were designed to amplify a726-bp fragment that covers the MYD88 L265P mutation (forward: 5′-gggata tgc tga act aag ttg cca c-3′(SEQ ID NO: 1) and reverse: 5′-gac gtgtct gtg aag ttg gca tct c-3′ (SEQ ID NO: 2)). Twenty nanograms ofgenomic DNA were used for PCR amplification. After initial denaturationat 95° C. for 2 minutes, 40 cycles of PCR amplification were performed,each consisting of a denaturing step of 94° C. for 30 seconds, annealingat 65° C. for 30 seconds, and extension at 68° C. for 1 minute, followedby a final step at 68° C. for 10 minutes. The amplified fragments wereisolated by QIAquick gel extraction kit (Qiagen, CA) and sequenced usingthe forward primer 5′-gct gtt gtt aac cct ggg gtt gaa g-3′ (SEQ ID NO:3), and the reverse primer 5′-gac gtg tct gtg aag ttg gca tct c-3′ (SEQID NO: 4).

Sanger sequencing was used to validate WGS results in tumor samples fromall 30 WM patients, and also in normal paired tissue from 19 patients,including the 10 individuals with paired WGS data. In as well, CD19+ andCD138+ isolated BM cells from an unrelated cohort of 12 other WMpatients; CD19+ isolated BM cells from 8 IgM MGUS patients; CD138+isolated BM cells from 8 multiple myeloma (MM) patients; CD19+ isolatedPB mononuclear cells from 12 healthy donors; as well as the BCWM.1,MWCL-1, and WM-WSU WM cell lines; the IgM secreting Ramos cell line, andthe MM1.S, RPMI 8226, and U266 MM cell lines were also Sanger sequencedfor MYD88. TA cloning and sequencing of at least 100 clones was alsoperformed using the above primers for CD19+ isolated BM cells for 4patients with IgM MGUS (Genewiz, S. Plainfield, N.J.).

Results

Tumor and normal genomes were both sequenced to an average of 66× (range60-91×) coverage of mapped individual reads. The average gross mappedyield for these genomes was 186.89 (range 171.56-262.03 Gb). In ourinitial analysis of the 10 paired genomes, we identified a recurringsequence variant with a variant score of 189 (range 74-345) at position38182641 in chromosome 3p22.2 which results in a single nucleotidechange from T→C in the myeloid differentiation primary response (MYD88)gene, and a predicted non-synonymous change at amino acid position 265from leucine to proline (L265P). This variant was the most common of amedian of 3,419 (range 2,540-4,011) somatic variants identified by WGSin the 10 paired patients using a somatic score of 0.1. By WGS, theMYD88 L265 variant was found as a somatic mutation in tumor cells fromall 10 paired patients, as it was not found in their matched normaltissues, and was also seen in tumor cells from 16 of the 20 unpaired WMpatients. The presence versus absence of the MYD88 L265 mutation inthese patients was not impacted by time from WM diagnosis (14.1 versus14.8 months, respectively; p=0.40). For 22 of 26 patients, the MYD88L265P variant was heterozygous, whereas in 4 patients an acquired UPDevent (median 49.5 MB, range 48.5-50.0 MB) at 3p22.2 resulted inhomozygous presence of the variant in at least a subset of tumor cells(FIGS. 1A, 1B). These UPD events were absent in normal tissue for the 2of 4 patients who had paired samples (FIGS. 1C, 1D). No distinguishingclinical and laboratory features for patients with homozygous versusheterozygous MYD88 L265 mutation were observed, though patients withhomozygous MYD88 L265 had a considerably longer interval from WMdiagnosis (56.4 versus 11.1 months; p=0.30), and invariably a largerpatient series will be required to determine the significance of thisfinding.

Sanger sequencing confirmed the presence of the MYD88 L265P variant inall 26 tumor samples revealed by WGS, as well as in one additionalpatient's tumor sample which had read level evidence of the MYD88 L265Pvariant but was not called with adequate confidence. In this patient, asubpopulation (approximately 10%) of tumor cells exhibited the MYD88L265P mutation. By Sanger sequencing, the L265P MYD88 variant wastherefore present in tumors of 27 of 30 (90%) WM patients, whichincluded patients with sporadic as well as familial disease in whom thevariant was observed at the same frequency. Sanger sequencing alsoconfirmed the absence of the MYD88 L265P mutation in normal tissue fromthe 10 paired patients who underwent WGS, as well as in 9 other unpairedpatients who had the MYD88 L265 mutation by WGS, and for whom normaltissue was available.

In an unrelated cohort of 12 WM patients, we further observed by Sangersequencing a heterozygous MYD88 L265P variant in 11 (92%) patients.Importantly, the MYD88 L265P variant was present in both CD19+ andCD138+ selected cells in these patients consistent with the knowndistribution of the malignant WM clone which extends from mature B-cellsto plasma cells. By Sanger sequencing, we also detected a heterozygousMYD88 L265P variant in BCWM.1 and MWCL-1 WM cells. In contrast, theMYD88 L265P variant was not found in the IgM secreting cell lines WM-WSUand Ramos, both of which carry t(8;14), and in none of the myeloma celllines. Furthermore, we did not detect the MYD88 L265P variant in CD19+selected B-cells from 12 healthy individuals, nor in tumor samples from8 of 8 myeloma patients. By direct Sanger sequencing, the MYD88 L265mutation was absent in 7 of 8 (87.5%) IgM MGUS patients whosecharacteristics are described in Table 2. In the sole IgM MGUS patientin whom the MYD88 L265P mutation was detected, increased LPC were notedin his BM aspirate, and a core biopsy showed an isolated nodule but nodistinct infiltration by LPL cells required for the consensus diagnosisof WM. His subsequent course has been marked by progressively rising IgMlevels, and a falling hematocrit. TA cloning and sequencing of at least100 clones was also undertaken for 4 IgM MGUS patients whose directSanger sequencing did not reveal the MYD88 L265 variant. The MYD88 L265variant was absent in all clones for these 4 IgM MGUS patients. Thesefindings may potentially signify that acquisition of the MYD88 L265variant represents a transforming event from IgM MGUS to WM, or that thefrequency of the MYD88 L265 variant is too low to be picked up in IgMMGUS patients by either WGS or Sanger sequencing. Another considerationis that IgM MGUS might itself be heterogeneous, with acquisition of theMYD88 L265P leading to WM, while in other cases with wild type MYD88, adifferent oncogenic trajectory may result.

The discovery of a mutation in MYD88 is of significance given its roleas an adaptor molecule in Toll-like receptor (TLR) and interleukin-1receptor (IL-1R) signaling. All of the TLRs except for TLR3 use MYD88 tofacilitate their signaling. Following TLR or IL-1R stimulation, MYD88 isrecruited to the activated receptor complex as a homodimer which thencomplexes with IRAK4, leading to its autophosphorylation. TheMYD88/IRAK4 complex then recruits and activates IRAK1 and IRAK2. Tumornecrosis factor receptor associated factor 6 (TRAF-6) is then activatedby IRAK1 leading to early NF-κB activation, whereas IRAK2 facilitateslate NF-κB activation. The L265P mutation in MYD88 occurs at a residuethat is highly conserved in evolution, and contributes to a (3-sheet atthe hydrophobic core of the domain. Staudt and colleagues recentlyreported the presence of the MYD88 L265P mutation in a subset of tumorstaken from patients with the ABC subtype of diffuse large B-celllymphoma (DLBCL), and in a few patients with gastric mucosa-associatedlymphoid tissue (MALT) lymphomas. In contrast, the MYD88 L265P mutationwas absent in tumor samples from patients with the GCB subtype of DLBCL,and Burkitt's lymphoma. The frequency of 90% observed for the MYD88 L265variant in our studies from 2 independent cohorts of WM patients is farmore extensive than that observed in DLBCL (29%) and MALT (6%)lymphomas.

Staudt and colleagues also demonstrated that ectopic expression of MYD88L265P, but not wild type MYD88, prevented apoptosis of L265P expressingABC DLBCL cell lines that underwent total MYD88 knock down.Co-immunoprecipitation experiments in these cell lines with the L265Pmutation also showed enhanced binding to and phosphorylation of IRAK1,which was absent in cells with wild type MYD88. Additionally, knock downstudies of MYD88 or IRAK1, or inhibition of IRAK1 function with anIRAK1/4 kinase inhibitor led to loss of NF-κB signaling. Similarly, wehave observed that knockdown of MYD88 by lentiviral transduction, and/ortreatment with a MYD88 homodimerization inhibitory peptide17 or IRAK1/4inhibitor18 of BCWM.1 and MWCL-1 WM cells which possess the MYD88 L265Pmutation leads to loss of NF-κB signaling and enhanced apoptosis. SinceNF-κB signaling represents a critical determinant of WM cell growth andsurvival, targeting of tonic MYD88/IRAK signaling is of potentialrelevance to WM therapy.

In summary, using WGS, a novel, widely expressed somatic variant (MYD88L265P) has been identified in malignant LPC of WM patients, which helpsto discriminate WM from other overlapping entities, and whose putativerole in NF-κB signaling is of relevance to WM lymphomagenesis. Thesestudies offer novel insights into the pathogenesis of WM, and provide aframework for the development of diagnostic tools and targeted therapiesfor patients with WM.

Example 2

Materials and Methods

Patient and Samples

Ninety seven patients meeting diagnostic criteria for WM and 40 healthydonors (10 BM and 30 PBMC) were included in this study. Theirparticipation was approved by the IRB at the Dana-Farber CancerInstitute. Bone marrow mononuclear cells (BMMC) were sorted usingmagnetic beads. Purity of isolated B-cells (CD19+) was over 90%. DNA wasextracted using Allprep DNA/RNA mini kit (Qiagen, Valencia, Calif.).DLBCL cell lines OCI-LY3 and OCI-LY19 were kindly provided by Dr. MarkMinden from University Health Network in Canada.

Allele-Specific Polymerase Chain Reaction (AS-PCR)

Two reverse primers were designed to differentiate the mutant andwild-type allele of MYD88 L265P. To optimize the specificity, aninternal mismatch in the third position from the 3′-end was introduced.The mutant-specific reverse primer was 5′-CCT TGT ACT TGA TGG GGA aCG-3′(SEQ ID NO: 5) and the wild-type-specific reverse primer was 5′-GCC TTGTAC TTG ATG GGG AaC A-3′ (SEQ ID NO: 6). The common forward primer was5′-AAT GTG TGC CAG GGG TAC TTA G-3′ (SEQ ID NO: 7). PCR reaction wasperformed in a final volume of 25 ul with 50 nM of each primer and 50 ngDNA using PCR SuperMix High Fidelity (Life technology, CA). Thermalcycling conditions were: 2 min at 94° C., followed by 40 cycles of 94°C. for 30 s, 57° C. for 30 s, and 68° C. for 30 s, with a finalextension at 68° C. for 5 min. The amplified PCR products (159-bp) wereseparated on 2% agarose gel. To confirm the sequence, PCR products werepurified by QIA quick gel extraction kit (Qiagen, CA) and sequencedusing both forward and reverse PCR primers.

Real-Time AS-PCR

Quantitative detection of the MYD88 L265P mutation was developed usingthe primers described above and Power SYBR® Green PCR Master Mixaccording to manufacturer's instruction on the ABI Prism 7500 SequenceDetection System (Applied Biosystems, Foster City, Calif.). Briefly, PCRreaction was performed in a final volume of 25 μl with 25 nM of eachprimer and 50 ng DNA. Thermal cycling conditions were: 10 min at 950 C,followed by 40 cycles of 95° C. for 15 s and 60° C. for 60 s. Eachsample was assayed in triplicate. The standard curve for MYD88 L265P wasgenerated by a serial dilution of the mutant DNA with the wild-type DNA(50%, 10%, 2%, 0.4%, 0.08%, and wild-type). For the correspondingreference PCR, the forward primer is same as the one used for the AS-PCR(5′-AAT GTG TGC CAG GGG TAC TTA G-3′; (SEQ ID NO: 7)) and the reverseprimer is located at 53-bp downstream of the AS-PCR primer (5′-TGG TGTAGT CGC AGA CAG TGA-3′; (SEQ ID NO: 8)). Levels of the mutant MYD88L265P in patient samples were calculated based on the value of delta CTand the standard curve.

Sanger Sequencing

The forward PCR primer 5′-GGG ATA TGC TGA ACT AAG TTG CCA C-3′ (SEQ IDNO: 1) and reverse PCR primer 5′-GAC GTG TCT GTG AAG TTG GCA TCT C-3′(SEQ ID NO: 4) were designed to amplify a 726-bp fragment covering theMYD88 L265P site. Amplified PCR products were isolated by QIA quick gelextraction kit (Qiagen, CA) and sequenced using the reverse PCR primerand a sequencing primer 5′-GCT GTT GTT AAC CCT GGG GTT GAA G-3′ (SEQ IDNO: 3).

Statistical Analysis

Correlation between the MYD88 L265P status and clinical parameters wasevaluated by non-parametric ANOVA. Correlation between the changes of BMinvolvement and levels of mutant MYD88 L265P was assessed by linearregression. All analyses were performed with R (R Foundation forStatistical Computing, Vienna, Austria).

Results

Conventional AS-PCR Design

The somatic mutation L265P in the MYD88 gene was found in approximately90% of WM patients. To efficiently and quickly determine the MYD88 L265Pstatus, an inexpensive AS-PCR assay was developed that can be easilyimplemented in any laboratories with conventional PCR technology. Asshown in FIG. 2A, the AS primers are located in exon 5 while the commonforward primer is located in intron 4. To enhance the specificity in theAS-PCR reaction, an additional mismatch (T>A) was introduced at thethird position from the 3′ end of the AS primers. Sensitivity of theAS-PCR assay was assessed by a serial dilution of the DNA isolated fromDLBCL cell line OCI-LY3 (homozygous MYD88 L265P) with the DNA from DLBCLcell line OCI-LY19 (wild-type MYD88 L265P). The MYD88 L265P status ofthe cell lines was confirmed by Sanger sequencing (FIG. 2B). Thesensitivity assessments demonstrated that the mutant allele of MYD88L265P can consistently be detected at a dilution of 0.1% in thegel-based AS-PCR assay (FIG. 2C).

This assay was then applied to 97 WM patients who had not receivedpharmacological interventions at the time of BM biopsies. 87/97 (89.7%)were found positive for the MYD88 L265P mutation. To further verify theAS-PCR assay, the entire cohort was sequenced for the MYD88 L265Pposition. Among the 87 patients positive for MYD88 L265P by AS-PCR, 82were positive while 5 were negative by Sanger sequencing. The 5 patientsnegative for MYD88 L265P by Sanger sequencing showed weak signals in thegel-based AS-PCR assay. By contrast, all patients negative for MYD88L265P by AS-PCR remained negative by Sanger sequencing. In addition, DNAfrom 40 healthy donors (10 BM and 30 PBMC) was analyzed and no MYD88L265P mutation was detected by either of the methods. The overallresults suggest that this AS-PCR assay is simple, reliable, andsensitive. In addition, Sanger sequencing analysis suggested that fivepatients carried homozygous MYD88 L265P because the mutant allele peakwas significantly higher than the wild-type allele peak. FIG. 2D showsan example of the homozygous carriers. This patient had 70% of BMinvolvement, so that the wild-type allele still can be detected bySanger sequencing. Homozygous MYD88 L265P could be caused by an acquireduniparental disomy (UPD) event at 3p22.2.

Quantitative Detection of MYD88 L265P Mutation

Next, a SYBR green-based real-time PCR was developed to allowquantification of MYD88 L265P. Given the high frequency of the MYD88L265P mutation in WM, quantitative assessment of MYD88 L265P haspotential to be developed as a robust biomarker for monitoring diseaseprogression and response to treatment. Sensitivity and specificity ofthe real-time AS-PCR were determined by a serial dilution of the mutantDNA with the wild-type DNA. C_(T) values were recorded formutant-specific and reference PCR and the corresponding delta C_(T)values were calculated. As shown in FIG. 3A, this real-time AS-PCR candetect the MYD88 L265P mutation at a dilution of 0.08% with more than 2cycle differences from the wild-type DNA background. Correlationcoefficient of the standard curve was 0.998 with a slope value of −3.5(FIG. 3B). The melting curve analysis revealed that the MYD88 L265Pmutant-specific amplicon melted at 840 C (FIG. 3C). A minor non-specificamplification was only found in the dilution of 0.4% or lower with amelting peak at 80° C.

To gain more information on the performance of the assays, all samplesthat have been analyzed by the gel-based AS-PCR and Sanger sequencingwere reanalyzed by the real-time AS-PCR. As shown in the scatter plot(FIG. 3D), the healthy donors (n=40) had delta CT values ranging from10.7 to 16.9 cycles (mean, 13.9 cycles; median, 14.1 cycles), whereasthe WM patients had delta CT values ranging from −0.2 to 15.2 cycles(mean, 3.3 cycles; median, 1.9 cycles). There were two major clusters ofWM patients separated by 3.2 cycles. The cluster with delta CT valuesranging from 9.6 to 15.2 cycles (n=10) looked similar to healthy donors.Thus, this cluster was determined as MYD88 L265P negative. By contrast,another cluster had delta CT values ranging from −0.2 to 6.4 cycles(n=87). This cluster was determined as MYD88 L265P positive. Inaddition, the gel-based AS-PCR and the real-time AS-PCR showed anexactly same result of determining the status of MYD88 L265P in thiscohort. The overall results suggest that a small subset of WM patientsmay not carry the MYD88 L265P mutation. Furthermore, the entire codingregion of the MYD88 gene was sequenced for the 10 patients who werenegative for MYD88 L265P as determined by AS-PCR. No MYD88 mutation wasfound.

Correlation Between MYD88 L265P Mutation Status and ClinicalCharacteristics

MYD88 L265P was initially reported in DLBCL as a gain-of-functionmutation that promotes NF-kB and JAK-STAT3 signaling through activatingthe IRAK family of serine-threonine kinases. Interestingly, MYD88 L265Pwas frequently mutated in the ABC subtype of DLBCL (29%) but rare in theGCB subtype. We sought to evaluate the differences of clinicalcharacteristics between the two clusters of WM patients determined byAS-PCR. Non-parametric ANOVA analysis revealed that the MYD88 L265Ppositive patients tended to have greater BM involvement (p=0.001), lowerserum IgG (p=0.011), and higher serum IgM (p=0.007) compared to theMYD88 L265P negative patients (Table 3). Due to small number of MYD88L265P negative patients, the observations need to be confirmed in anindependent study with large sample size.

Determination of Therapeutic Effect by Quantitative Assessments of MYD88L265P

To explore the potential of using the real-time AS-PCR method todetermine therapeutic effect and monitor residual disease in WM,concordance between the readouts of BM involvement and levels of mutantMYD88 L265P for seven patients who received pharmacological interventionand provided BM biopsies before and after treatment was examined. Theresults are summarized in Table 4. Patient A was a homozygous carrier ofMYD88 L265P and had 70% BM involvement and high level of mutant MYD88L265P before treatment. This patient exhibited a complete clinicalremission after the treatment. Accordingly, the BM involvement and thelevels of mutant MYD88 L265P became undetectable. Patient B and C showedapproximately 90% decrease in the BM involvement with the treatments.Similarly, a marked decrease in the levels of mutant MYD88 L265P wasalso observed from the two patients (96% and 74%, respectively). PatientD showed 47% decrease in the BM involvement versus 45% decrease in thelevels of mutant MYD88 L265P. However, the levels of mutant MYD88 L265Pwere little changed in patient E and F who showed 17% and 37% decreasesin the BM involvement, respectively. The last patient (G) did not appearto respond to the treatment and showed slightly increase in both BMinvolvement and levels of mutant MYD88 L265P. Linear regression analysisrevealed a high correlation between the percentage changes of BMinvolvement and levels of mutant MYD88 L265P (R2=0.90, p=0.001).Although the study sample size was very small, the results supported thehypothesis of using quantitative assessment of MYD88 L265P to determinetherapeutic effect and monitor residual disease in WM.

BM Examination is Essential in the Clinical Staging of Non-Hodgkin'sLymphomas (NHLs)

Merli et al reported a comparison between histology and flow cytometry(FC) on the assessments of BM involvement in non-Hodgkin's lymphomas(NHLs) including LPL (Assessment of bone marrow involvement innon-Hodgkin's lymphomas: comparison between histology and flowcytometry, Eur J Haematol, 85(5):405-15, 2010.). A high concordancebetween the two methods was observed for most NHLs. However, significantdiscordance was reported in LPL. It was speculated that FC mayunderestimate the extent of infiltrate with respect to histology.Quantitative assessment of MYD88 L265P in BM biopsy represents a noveltool contributing to clinical staging at diagnosis in WM.

In conclusion, a sensitive and inexpensive real-time AS-PCR method hasbeen developed that permits reliable and quantitative assessments ofMYD88 L265P. This is the first quantitative assay for MYD88 L265P andwill facilitate testing the potential of MYD88 L265P as a biomarker toimprove diagnosis and clinical staging and monitor disease progressionand response to treatment in WM.

Example 3

Bruton's tyrosine kinase (BTK) promotes B-cell receptor signaling alongwith B-cell expansion and survival through NF-κB and MAPK. MYD88 L265Pis a widely expressed somatic mutation in tumor cells from WM patients.MYD88 L265P promotes enhanced tumor cell survival through IRAK 1/4mediated NF-κB and MAPK signaling. We therefore sought to clarify therole of BTK signaling in MYD88 L265P expressing WM cells, and the impactof BTK and MYD88/IRAK inhibition on WM cell signaling and survival.

Materials and Methods

Western blot analysis was performed using total and phospho-specificantibodies in MYD88 L265P expressing WM cells, BCWM.1 and MCWL-1following MYD88 knockdown by lentiviral transduction, and/or use ofMYD88 or IRAK signal inhibitors. Cells were also treated with the BTKinhibitor PCI-32765, in the presence or absence of MYD88homodimerization or IRAK1/4 inhibitors. Annexin V/PI staining was usedto assess cell survival, and synergism assessed with CalcuSyn software.

Results

BTK was highly expressed and phosphorylated in MYD88-L265P expressing WMcells and PCI-32765 significantly blocked the BTK activation (FIG. 4).Increased phosphorylation of BTK was confirmed by western blotting withphospho-specific antibody in Waldenstrom's Macroglobulinemia (WM) celllines, BCWM.1 and MWCL-1, compared to Multiple myeloma cell lines, ANBL6and INA6. Antibody against total BTK was used as loading control.PCI-32765 significantly blocked the BTK phosphorylation in WM cells.

PCI32765 significantly reduced downstream NF-kB, MAPK and STAT3signaling in WM cells (FIG. 5). In addition to significantly blockingthe BTK activation, PCI-32765 also blocked the downstream NF-kB, MAPK,Stat3 signaling by significantly reduced the phosphorylation of IKBα,ERK1/2 and Stat3 proteins in WM cell lines, BCWM.1 and MWCL-1, comparedto multiple myeloma cell lines, ANBL6 and INA6. Antibodies againstcorresponding total proteins and GAPDH were used as loading controls.

Knockdown of MYD88 by lentiviral transduction, and/or use of a MYD88inhibitor leads to decreased BTK phosphorylation (FIG. 6). MYD88knockdown was confirmed by western blot in BCWM.1 and MWCL-1 cells. Theknockdown of MYD88 reduced BTK phosphorylation compared with controls.MYD88 homodimerization inhibitory peptides significantly reduced BTKphosphorylation compared with control peptides. Antibodies against totalBTK and/or GAPDH were used as loading control.

Treatment with PCI-32765 induces apoptosis of MYD88 L265P expressing WMcells (FIG. 7A). PCI-32765 shows robust tumor cell killing incombination with a MYD88 pathway inhibitor in primary WM patients bonemarrow tumor cells (FIG. 7B). PCI-32765 shows synergistic tumor cellkilling in combination with an IRAK 1/4 kinase inhibitor (FIG. 7C).

BTK activation is facilitated by MYD88 pathway signaling in MYD88 L265Pexpressing WM cells, and participates in MYD88 downstream signaling.Inhibition of BTK by PCI-32765 leads to robust tumor killing of MYD88L265P expressing WM cells, which is potentiated by MYD88 pathwayinhibitors.

REFERENCES

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TABLE 1 Clinical and laboratory characteristics for 30 WM patients whoselymphoplasmacytic cells were used in whole genome sequencing studies.Median Range Age 62 41-87 Familial WM 9 (30%) NA Untreated 26 (87%) NABone Marrow Involvement (%) 65% 5-95% Clonal B-cells(%)¹ 96.3 55.5-100IgM (mg/dL) 3,970 590-6,580 IgMκ/IgMλ monoclonal protein 24/6 NA IgA(mg/dL) 41 11-516 IgG (mg/dL) 519 215-3,120 Serum B₂M (mg/L) 3.551.50-12.1 Hematocrit (%) 31.4 24.0-41.5 ¹Determined by light chainrestriction using flow cytometry.

TABLE 2 Clinical and laboratory characteristics for the 8 IgM MGUSpatients who underwent Sanger sequencing for the MYD88 L265P variant.Median Range Age 62 52-71 Familial WM 2 (25%) NA Untreated 8 (100%) NABone Marrow Involvement (%) 0% 0-2% Clonal B-cells(%)¹ 0% 0-1 IgM(mg/dL) 682 142-1,190 IgMκ/IgMλ monoclonal protein 5/3 NA IgA (mg/dL)142 53-244 IgG (mg/dL) 772 523-1,040 Serum B₂M (mg/L) 1.70 1.30-2.10Hematocrit (%) 41.4 36.7-44.8 ¹Determined by light chain restrictionusing flow cytometry.

TABLE 3 Correlation between MYD88 L265P mutation status and clinicalcharacteristics MYD88 L265P mutation status Clinical parameter PositiveNegative p-value* Age at diagnosis n = 87 60.26 n = 10 66.50 0.064(9.97) (8.33) Gender, % female n = 86 40.91% n = 10 50.00% 0.738** Bonemarrow n = 85 52.92 n = 10 18.00 0.001 involvement, % (30.49) (18.74)IgA, mg/dL n = 85 622.79 n = 10 970.10 0.106 (552.45) (711.69) IgG,mg/dL n = 85 216.35 n = 10 332.10 0.011 (322.36) (284.60) IgM, mg/dL n =85 3093.62 n = 10 1536.20 0.007 (1943.89) (2039.79) Values are mean(SD). *Non-parametric ANOVA **Fisher's exact test

TABLE 4 Concordance between the assessments of BM involvement and MYD88L265P mutation levels. Levels % of change mutant % of BM MYD88 changeMYD88 Age at Treatment Involvement L265P of BM L265P Patient diagnosisGender status (%) (%)* involvement levels A** 61 Male Untreated 70156.90 100 100 Bendamustine Negative Negative rituxan B 44 MaleUntreated 90 60.73 89 96.33 R-CD 10 2.23 C 52 Male Untreated 50 78.12 9073.61 R-CD 5 19.08 D 59 Male Untreated 95 99.15 47 45.39 Everolimus 5054.15 E 63 Male Untreated 90 96.07 17 1.14 Everolimus 75 94.97 F 70 MaleUntreated 95 95.93 37 8.61 Everolimus 60 87.67 G 63 Male Untreated 2067.93 −25 −12.87 Everolimus 25 76.67 R-CD: combination of rituximab,cyclophosphamide, and dexamethasone. BM biopsy was collected before andafter treatment. % of mutation was calculated based on standard curve.**Homozygous carrier of the MYD88 L265P mutation.

1-16. (canceled)
 17. A method to treat lymphoplasmacytic lymphoma in asubject, the method comprising: selecting a subject on the basis thatthe subject has a mutation at position 38182641 in chromosome 3p22.2,administering to the subject a myeloid differentiation primary response88 (MYD88) inhibitor, an interleukin receptor associate kinase 1/4(IRAK-1/4) inhibitor, and/or a Bruton's tyrosine kinase (BTK) inhibitorin an amount effective to treat lymphoplasmacytic lymphoma.
 18. Themethod of claim 17, wherein the biological sample is a sample of bonemarrow, lymph node, spleen or blood.
 19. The method of claim 17, whereinthe mutation results in a single nucleotide change from T to C in themyeloid differentiation primary response 88 (MYD88) gene.
 20. The methodof claim 17, wherein the mutation results in an amino acid change fromleucine to proline at position 265 in the myeloid differentiationprimary response 88 protein.
 21. The method of claim 17, wherein theMYD88 inhibitor is a peptidomimetic compound ST2825.
 22. The method ofclaim 17, wherein the IRAK-1/4 inhibitor isN-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole.
 23. Themethod of claim 17, wherein the BTK inhibitor is Ibrutinib (PCI-32765).24. A method for monitoring progression or recurrence oflymphoplasmacytic lymphoma (LPL) in a subject, comprising obtainingmultiple biological samples of a subject over a period of time,determining from the multiple biological samples the level of atranscript comprising a mutation at position 38182641 in chromosome3p22.2, wherein a change in the level of the transcript over the periodof time is indicative of the progression or recurrence of LPL in thesubject.
 25. The method of claim 24, wherein the subject is undergoingchemotherapy.
 26. The method of claim 24, wherein the level of thetranscript is measured using quantitative real time polymerase chainreaction.
 27. The method of claim 26, wherein the quantitative real timepolymerase chain reaction is performed using an allele specific primer,wherein the allele specific primer hybridizes at or near its 3′ end tothe mutation at position 38182641 in chromosome 3p22.2.
 28. The methodof claim 27, wherein the allele specific primer is SEQ ID NO:
 5. 29. Themethod of claim 24, wherein the biological sample is a sample of bonemarrow, lymph node, spleen or blood.
 30. The method of claim 24, whereinthe mutation results in a single nucleotide change from T to C in themyeloid differentiation primary response 88 (MYD88) gene.
 31. The methodof claim 24, wherein the mutation results in an amino acid change fromleucine to proline at position 265 in the myeloid differentiationprimary response 88 protein. 32-35. (canceled)
 36. A method forfacilitating the diagnosis of lymphoplasmacytic lymphoma in a subject,the method comprising: selecting a subject on the basis that the subjectpresents one or more of the following clinical features: anemia,hyper-viscosity, neuropathy, coagulopathies, splenomegaly, hepatomegaly,adenopathy, and an IgM serum paraprotein, obtaining a biological sampleof the subject, determining from the biological sample whether thesubject has a mutation at position 38182641 in chromosome 3p22.2, andproviding a report summarizing statistically significant resultsindicating that the subject has lymphoplasmacytic lymphoma if thesubject has the mutation.